Methods for producing human antibodies in SCID mice

ABSTRACT

An improved method for producing human antibodies in SCID mice is provided. The improvement includes the use of dendritic cells pulsed with antigen-antibody complexes and antigen-antibody complexes as immunizing agents.

FIELD OF THE INVENTION

The subject invention provides a novel and reproducible method forproducing human monoclonal antibodies to desired antigens, e.g. prostatespecific antigen. These monoclonal antibodies, because of their humanorigin, should be useful therapeutic agents, e.g. for the treatment ofhuman prostate cancer.

BACKGROUND OF THE INVENTION

Antibodies (Ab) that recognize and adhere to proteins on the surface ofbacteria, virus or parasites help immune system cells identify, attackand remove them from the body. Similarly, monoclonal Ab (MoAb) thatadhere to cancer cells but not to normal cells can be an effectivetherapy for human cancers. Such MoAbs are generally murine Absgenetically modified to contain human constant regions (“humanized”).However, fully human MoAb are potentially superior to humanized murineMoAb as therapies for human cancer because of their absence ofimmunogenicity in humans. Human B cells can be stimulated to produce Absthat recognize specific human target proteins. However, previous methodsare typically very complex and yield inconsistent results. Therefore,there exists a need in the art for improved methods for producing humanmonoclonal antibodies.

OBJECTS OF THE INVENTION

It is an object of the invention to obviate the problems of the priorart.

It is a specific object of the invention to provide a novel method forproducing human antibodies in SCID mice.

It is an even more specific object of the invention to provide a novelmethod for producing human antibodies in SCID mice wherein theimmunizing protocol includes the administration of dendritic cells whichhave been pulsed in vitro with antigen-antibody complexes and/orantigen-antibody complexes.

It is a more specific object of the invention to provide a novel methodfor producing human antibodies specific to human prostate specificantigen (PSA).

It is an even more specific object of the invention to provide a novelmethod for producing human antibodies to human PSA in SCID mice whereinthe immunization protocol includes the administration of dendritic cellswhich have been pulsed in vitro with PSA-anti-PSA antibody complexesand/or PSA-anti-PSA antibody complexes.

It is still another object of the invention to provide a novelimmunization protocol for producing human antibodies in SCID mice thatincludes in vivo transformation with EBV during immunization.

BRIEF DESCRIPTION OF THE INVENTION

As discussed in greater detail infra, by judicious experimentation, thepresent inventors have developed an improved method for producing humanantibodies in SCID mice. Specifically, it has been found thatimmunization of SCID mice with autologous dendritic cells, e.g.,autologous peripheral blood dendritic cells that have been pulsed invitro with a desired antigen, more preferably an antigen-antibodycomplex, yields high antibody titers wherein such antibodies possess thedesired specificity.

Also, it has been found that immunization with antigen-antibodycomplexes yields improved results, i.e., high serum antibody titerswherein such antibodies exhibit the desired specificity.

Still further, the present invention provides in particular a novelimmunization protocol for producing human monoclonal antibodies toprostate specific antigen (PSA). These antibodies, because of theirspecificity and human origin, should be useful for the treatment ofprostate cancer. Because of their human origin, they should possesshuman antibody effector functions and should elicit no immunogenicity.

DESCRIPTION OF THE FIGURES

FIG. 1 schematically depicts the immunization strategy of the invention.

FIG. 2 is a flow chart summarizing the engraftment and immunization ofSCIDhu PBL mice.

FIG. 3 is a FACS analysis of peripheral blood dendritic cells culturedin serum free media. DC were grown in triplicate cultures, harvested onday 7, pooled and subjected to FACS analysis as described in “Materialsand Methods”. The DC generated from PBMC used to reconstitute the SCIDhuPBL mice were 65% large, MHC class II⁺/CD33⁺/CD40⁺/CD1a^(lo)/CD14⁻ cellswith dendritic morphology. The remaining cells were mostly T cells andsome B cells. These results are similar to those obtained from culturesgenerated from 8 individual PBMC donors. All donors generated culturesthat were between 50 and 75%CD11c^(hi)/CD32⁺/CD33⁺/CD40⁺/CD45RO⁺/−ClassII⁺/B7.1⁺/B7.2⁺DC. DCgenerated from different donors were heterogeneous for CD1a, CD4, CD14,and CD64 expression (Data not shown).

FIG. 4 is a comparison of MHC and T cell co-stimulatory surface Agexpression by DC cultures. DC were grown in triplicate cultures,harvested on day 7, pooled and subjected to FACS analysis as describedin “Materials and Methods”. Results show MHC class II, B7.1, B7.2 andCD40 expression was significantly enhanced on DC pulsed with soluble PSAbut not PSA-mIG_(2a). Similar results were obtained with DC culturesgenerated from another donor and pulsed with Tetanus toxoid.

FIG. 5 is a quantitation of human IgG in sera of SCIDhu PBL mice. Serawas collected on days 14 and 28. Total and PSA specific human IgG werequantitated by ELISA. Results shown are the average of 8 mice per group.Error bars represent ±Std. Dev. A. Total human IgG (mg/ml). Group H miceIgG sera concentrations ranged between 0.56 and 2.19 mg/ml IgG by day28. IgG sera concentrations in groups F and G control mice rangedbetween 8 and 840 μg/ml IgG by day 28. B. PSA specific IgG (μg/ml). PSAspecific IgG was quantitated using a mouse monoclonal IgG specific forhuman PSA as a standard. Only Group H produced PSA specific IgG. C.Percent PSA specific IgG. The relative quantity of PSA specific IgG wascalculated as follows: [PSA specific IgG]/[total IgG]×100.

FIG. 6 is an analysis of PSA specificity of group H sera IgG. A.Relative PSA specificity. Human IgG in group H sera binds PSA ten timesgreater than the nonspecific binding generated by group F sera with anequivalent concentration of IgG or by an equivalent concentration ofpurified human IgG. B. Soluble PSA competition ELISA. Soluble PSAinhibits the binding of group H sera IgG in a concentration dependentmanner.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have developed a novel and reproducible method tostimulate human B cells to make Ab that adhere to desired antigens, e.g.prostate specific antigen (PSA), a protein on the surface of prostatecancer cells. Using these methods, specific human monoclonal antibodiesto desired antigens can be cloned, which have applicability in humantreatments, e.g., the treatment of prostate cancer.

The advantages of the subject invention are significant.

In particular, these methods are advantageous for the rapid productionof fully human monoclonal antibodies for immunotherapy of humandiseases.

The major distinguishing differences of the subject protocols comparedto prior practices are the use of Ab-antigen (Ag) complexes andautologous dendritic cells (DC) as immunizing adjuvants.

Still another non-obvious distinguishing difference of the subjectmethods in relation to previous methods is the inclusion of intentionalEBV transformation in vivo during the unique DC/Ab-Ag compleximmunization steps. Also, the present inventors have determined optimalconditions for Ag boosting SCIDhu PBL mice (using PSA as a modelantigen), and high affinity antibodies to PSA using two differentdonors.

In order to generate human anti-PSA specific IgG responses that could beimmortalized we have developed a novel SCIDhu mouse immunizationprotocol (see FIG. 1). See, also, FIG. 2, which is a flow chartsummarizing the engraftment and immunization of SCIDhu mice. Briefly, wehoped that complexing antigen to the Fc receptors of dendritic cells(DC) would increase the immunogenicity of the antigen. (However, giventhe inherent unpredictability with monoclonal antibody manufacture, thisresult was not assured.) Therefore, we pulsed dendritic cells isolatedand expanded from a particular donor with PSA complexed to a mouseanti-PSA IgG_(2a) monoclonal Ab (Ab-PSA complex).

On day 0, SCID's were reconstituted with 10⁸ female PBL's and immunizedwith 25 mg Ab-PSA complex. Simultaneously, autologous, DC cultures wereinitiated. The DC were pulsed with 25 mg/ml Ab-PSA complex on day 6, andinjected i.p. on day 7. The mice were boosted with 25 mg of Ab-PSAcomplex on days 7 and 14 and with 25 mg of soluble PSA on day 21. Miceimmunized by this method generated PSA specific IgG sera concentrationsthat were comparable to those induced to Tetanus Toxoid using standardimmunization methods (TT in alum). Moreover, these results werereproduced in two separate experiments using different PBMC donors.Therefore, the present immunization protocol is reproducible andtherefore should be applicable to different antigens, in particularthose involved in human diseases.

As noted, the subject method uses dendritic cells which have been pulsedin vitro with antigen or antigen-antibody complexes as immunizingagents. Dendritic cells (DC) are professional antigen presenting cells(APC) that initiate immune response (see 1, 2 for review). Recently,several methods have been developed to generate human DC from peripheralblood mononuclear cell (PBMC) derived progenitor cells), ill vitro.These different culture methods yield several DC subtypes withheterogeneous morphology, phenotype and function. However, all of theseDC subtypes have been shown to be potent stimulators of naive Agspecific T cells (3-5). This is due in large part to the fact that DCexpress class I and II MHC and co-stimulatory cell surface moleculesB7.1 and B7.2 (6, 7). In addition, human DC pulsed with weaklyimmunogenic, tumor associated antigens (TAA) are capable of stimulatingTAA specific cytotoxic T lymphocyte (CTL) proliferation andcytotoxicity, in vitro, thus illustrating both their potency as APC andtheir potential utility as tumor specific vaccines (8-10).

DC derived from PBMC and cultured with GM-CSF and IL-4 express both thehigh affinity IgG receptor FcγRI (CD64) and the low affinity IgGreceptor FcτRII (CD32) at varying levels (3, 11). Both CD64 and CD32have been shown to mediate uptake of Ag by DC (11, 12). Targeting Ag toFcγR on human monocytes and DC via monoclonal antibody (mAb)-Agcomplexes reduces the amount of Ag required for Ag specific T cellactivation as much as 1000-fold (13, 14).

Although many recent studies have analyzed human T cell activation byDC, what is not clear is whether in vitro generated DC are capable ofstimulating a primary humoral immune response. DC isolated from mousespleen and pulsed with myoglobin were capable of stimulating a primaryhumoral immune response in syngeneic mice, but mouse splenic DC may havedifferent immunostimulatory effector functions than DC derived fromhuman peripheral blood (15). Follicular DC (FDC)-lymphocyte clustersisolated from human tonsil enhanced growth and Ig production by CD40activated human B cells, in vitro (16). However, tonsillar FDC arephenotypically and morphologically distinct from peripheral bloodderived DC and, therefore, are likely to have different effectorfunctions as well (17).

SCID mice are deficient in mature lymphocytes, Ig production andlymphocyte mediated immune responses due to defective Ig and T cellreceptor gene rearrangement (18). SCID mice reconstituted with humanperipheral blood lymphocytes (SCIDhu PBL mice) can be effective modelsof recall antigen directed Ig production by human B cells (19, 20).However, it is very difficult to stimulate neo-Ag, self-Ag or TAAspecific primary immune response and IgG production in SCIDhu PBL mice(21).

In this study we characterized the phenotype of DC generated from PBMCin low protein, serum free media. We then assessed the ability of serumfree cultured DC to stimulate a prostate specific antigen (PSA)specific, primary humoral immune response by SCIDhu PBL mice. We showedthat DC pulsed with PSA complexed to a mouse IgG_(2a) specific for humanPSA (PSA-mIgG_(2a)) can induce PSA specific human IgG production inSCIDhu PBL mice. SCIDhu PBL mice immunized with soluble PSA pulsed DCdid not produce PSA specific IgG. These results suggest that themechanism by which DC acquired Ag altered DC expression andimmunostimulating effector functions. Different Ag acquisitionmechanisms yield different co-stimulating molecule surface expressionand subsequent immunostimulatory effector functions by DC.

EXAMPLE

The following materials and methods were used.

DC Generation in Serum Free Cultures

PBMC were obtained from healthy donors by leukophoresis or byvenapuncture into heparinized tubes. RBC were removed from residual PBMCby hypotonic lysis in Gey's lysis buffer prior to freezing in 50% humanserum, 40% Iscoves complete media (Iscove's modified Delbucco's media(Irvine Scientific, Santa Ana, Calif.) plus sodium pyruvate, minimalessential amino acids, L-glutamine (Sigma, St. Louis, Mo.) andgentamicin (Gibco BRL, Grand Island, N.Y.)) and 10% DMSO (Sigma). FrozenPBMC were stored in LN₂. DC were grown essentially as described byRomani et al, except that Iscove's complete was supplemented with 2%Nutridoma® HU (Boehringer Mannheim Corporation, Indianapolis, Ind.)instead of 10% fetal bovine serum. Freshly isolated and thawed PBMC werepurified by Histopaquel (Sigma) gradient separation, washed and platedat 5×10⁶ cells/ml in IN2 at 37° C. for 2 hrs. Non-adherent cells weregently removed with the media, additional 37° C. IN2 was added and thecells were incubated at 37° C. for 5 additional minutes. Non-adherentcells were again gently removed and the residual cells were cultured inIN2 supplemented with 500 U/ml IL-4 and 800 U/ml GM-CSF (Genzyme, Inc.,Cambridge, Mass.). Cultures were fed with additional cytokines on day 3.Human PSA specific mouse monoclonal IgG₂a (Clone 10-P20; FitzgeraldIndustries International Inc., Concord, Mass.) was complexed with >99%pure PSA (Fitzgerald Industries International) at equimolar ratios at 4°C. overnight (PSA-mIgG_(2a)). The DC enriched cultures were pulsed with25 μg/ml (final concentration) PSA, PSA-mIgG_(2a) or an equivalentvolume of IN2 on day 6 and non-adherent cells were harvested on day 7.

Flow Cytometric Analysis

The following FITC and PE labeled monoclonal antibodies (mAb) were used:anti-HLA DR, DP, DQ, anti-CD 1a, anti-CD3, anti-CD11c, anti-CD 16,anti-CD32w (FcγRII), anti-CD33, anti-CD40, anti-CD45RO, anti-CD64(FcγRI), anti-CD86 (B7.2), (Pharmingen, San Diego, Calif.), anti-CD4,anti-CD 14, anti-CD80 (B7.1), PE-labeled isotype controls (Becton andDickinson, San Jose, Calif.), anti-ABC, and FITC labeled isotypecontrols (Harlan Bioproducts for Science, Inc., Indianapolis, Ind.). Day7 DC enriched cultures and single cell isolates from SCIDhu PBL mousetissues were washed and resuspended in 4° C. FACS buffer (1% BSA, 1×PBS,0.1% Na Azide and 40 μg/ml human IgG) at 1×10⁶ cells/ml. The cells werethen aliquoted and stained for 45 minutes with mAb diluted to themanufacturers' recommended concentration. The cells were washed twice inFACS buffer and data was acquired on a FACScan® (Becton Dickinson). Datawas analyzed using Lysis 1® ((Becton Dickinson) or F cap List® (SoftFlowHungary, Inc., Pecs, Hungary) software. Specific reactivity datashown as ΔMFI is calculated as follows: MFI of FITC or PE labeledspecific mAb—MFI of isotype and fluorochrome matched mAb control. Theseresults are contained in FIGS. 3 and 4.

SCID Mouse Engraftment and Immunization

PBMC were obtained from healthy female donors by leukophoresis. RBC wereremoved by hypotonic lysis in Gey's lysis solution. Residual PBMC werefrozen and stored as described above. Four to six week old male FoxChase ICR SCID™ mice (Taconic, Germantown, N.Y.) were housed, fed andhandled according to established protocols for immunodeficient strains.Mice were engrafted with 10⁸ PBMC, i.p., on day 0. Autologous DCcultures were initiated on day 0 as described above. Group F mice wereimmunized with 25 μg of PSA-mIgG_(2a) complex weekly, 7×10⁶ thawedautologous PBMC on day 7 and then boosted with 25 μg PSA on day 21.Group G Mice were immunized with 25 μg of soluble PSA weekly and 7×10⁶PSA pulsed DC enriched cells on day 7. Group H mice were immunized with25 μg of PSA-IgG_(2a) complex weekly, 7.5×10⁶ PSA-mIgG_(2a) pulsed DCenriched cells on day 7 and then boosted with 25 μg PSA on day 21. Serawas collected on days 14 and 28. Mice were sacrificed and spleens andlymph nodes were collected on day 28. Some spleens were laterallybisected and single cells isolated from one half were analyzed by flowcytometry as described above. The remaining spleens and LN were embeddedin OCT compound (Sukura Finetek, Inc., Torrance, Calif.) and thensimultaneously frozen and fixed in LN₂ chilled 2-methylbutane (Sigma)for immunohistochemical staining.

ELISAs

Human Ig sera concentrations were assayed by quantitative ELISAs. ELISAswere performed in 96 well Immulon 2′ “U” ELISA plates (DynatechLaboratories, Inc., Chantilly, Va.). Human IgG and IgM ELISA plates werecoated with 2 μg/ml polyclonal goat anti-human IgG or goat anti-humanIgM (Southern Biotechnology Associates, Inc., Birmingham, Ala.) inbicarbonate buffer (pH 9.3) overnight. PSA specific IgG plates werecoated with 99% pure PSA at 4 μg/ml in bicarbonate buffer. PSA specificIgG was quantitated using a mouse monoclonal IgG₁ specific for PSA(clone ERPR8, ICN, Costa Mesa, Calif.) as a standard. Incubations weredone at RT in serially diluted duplicate wells. Binding of Ig wasdetected by horseradish peroxidase (HRP) conjugated polyclonal goatanti-human IgM-HRP, polyclonal goat anti-human IgG-HRP or polyclonalgoat anti-mouse IgG-HRP secondary antibody (Southern BiotechnologyAssociates) incubation and subsequent enzymatic development ofo-phenylenediamine dihydrochloride (Sigma) substrate. Reactions werequenched with 4N H₂SO₄ and the plates were read on a ELISA plate readerat OD₄₉₀. The concentration of human Ig in SCIDhu PBL sera wasquantitated by comparison of SCIDhu PBL serum OD₄₉₀ values with seriallydiluted standard curves. These results are contained in FIG. 5.

To confirm the PSA binding specificity of group H sera IgG pooled serafrom four group H mice was diluted 1:15 (50 μg/ml total IgG finalconcentration) and 1:20 (50 μg/ml total IgG final concentration) intotriplicate wells containing serially diluted concentrations of solublePSA. Soluble PSA induced inhibition of PSA specific binding by group Hsera and by an equivalent concentration of control human IgG (Zymed,Inc.) was assayed using polyclonal goat anti-human IgG-HRP, as describedabove. These ELISA results are contained in FIG. 6.

Immunohistochemistry Analysis

Histologic and human lymphocyte specific antibody (CD3 and CD 19)staining of frozen and fixed SCIDhu PBL mouse tissues was contracted toBioPharMaceutical Support Services (Pharmingen).

Immunoblot Analysis

These experiments are ongoing.

Example 1

Human IgG Production in DC/Ab-PSA Complex Immunized

SCIDhu PBL Mice Mouse Monoclonal IgG.

Antibody (Cat. No. 10-P2O; Fitzgerald Industries Inc.) was complexedwith PSA at equimolar ratios at 4° C. over night (Ab-PSA complex) andthen dialyzed to remove azide. Autologous peripheral blood dendriticcells (pDC) were grown in serum free media and pulsed with either 25μg/ml soluble PSA or Ab-PSA complex. All mice received 10⁸ PBMC i.p. onday 0. Each group consisted of 8 mice. Group F mice were immunized with25 g of Ab-PSA complex weekly and with 25 μg PSA on Day 21. Group G micewere immunized with 25 μg of soluble PSA weekly and 7×10⁶ soluble PSApulsed pDC on day 7. Group H mice were immunized with 25 μg of Ab-PSAcomplex weekly, 7.5×10⁶ Ab-PSA pulsed pDC on day 7 and then 25 μg PSA onDay 21. These results are summarized in FIG. 6. The Day 14 results, (A)Graphs, from left to right are as follows: Average PSA specific IgG; PSAspecific IgG for individual mice; Average total human IgG; Average ofpercent specific IgG (specific IgG/total IgG×100); percent specific IgGfor individual mice. B. Day 28 results. Graphs, from left to right areas in A. All sera IgG concentrations are expressed as mg/ml. Error barsrepresent ±Std. Dev.

The immunization method described in FIG. 6 enhanced human lymphocyteengraftment in SCIDhu PBL mice. On the average, six times more human Tcells were detected per spleen and more enlarged lymph nodes (LN) wereisolated from group H mice than from either control group (see Table,below). Importantly, the enhanced engraftment and Ig production was notinduced at the expense of enhanced xenogeneic graft versus host disease(XGVHD), as has been reported in other “enhanced” SCIDhu systems. TABLESummary of Engraftment Total Spleen % hCD3+ % hCD19+ #Mice w. GroupCells Number* Cells{circumflex over ( )} Cells{circumflex over ( )}LN/group F 1.69 ± 0.26 × 10⁸ 2.30 ± 0.67 <2% 2/8 G 1.58 ± 0.32 × 10⁸2.93 ± 0.34 <2% 4/8 H 3.04 ± 0.60 × 10⁸ 8.93 ± 4.32 <2% 7/8

Mice immunized, as described in FIG. 6, were sacrificed on day 28.Spleens and LN were collected from all mice. Spleens were divided inhalf. One half was used to determine cell numbers and to do FACSanalysis (3 mice each group). The other half of the spleens and all theisolated LN were fixed and frozen for histologic analysis. Spleen cellnumber and percent human T cells are shown ±Std Dev. (p>0.5). *: averageof 8 Mice.ˆ: average of 3 mice.

Histologic antibody staining data (frozen/fixed slides were stained witha-hCD3 and a-hCD19) showed that Group H mice had many more human T and Bcells in enlarged peripheral LN compared to control mice. Also, spleensfrom group H mice had more localized B cell engraftment than controlmice (data not shown).

The specificity of the PSA IgG response by group H mice was confirmed Iby comparison with non-responding Group F sera, control human IgG and bycompetition of sera binding by soluble PSA.

Example 2

Specificity of Antibody Responses Obtained in Ab-PSA/pDC ImmunizedSCIDhu PBC Mice.

In this Example, the relative PSA specificity of pooled sera from groupH mice, control hIgG and group F serum is measured and was shown in FIG.5.

FIG. 5 shows relative PSA specificity of pooled sera from group H mice,control hihg and group F serum. In this Figure, open circles representpooled group H serum, and closed circles purified hIgG. The opentriangles represent mouse F1.1 serum. The data shows that group H serumbinds PSA 10 times greater than equal concentrations of either controlhIgG. Panel B shows the inhibition of group H sera specific binding bysoluble PSA. The open circles represent pooled group H serum diluted1:15 (120 μg/ml). The closed circles represent pooled group H serumdiluted 1;20 (90 μg/ml). The open tri-angles represent purified hIgGcontrol, (90 μg/ml). The data shows that pooled group H sera binding canbe inhibited by soluble PSA to OD₄₉₀ values obtained by an equalconcentration of control hIgG in et dose dependent manner.

CONCLUSIONS

Therefore, these results demonstrate that DC pulsed withantigen-antibody complexes induced PSA specific Ab responses in SCIDhuPBL mice better than previous immunization protocols. Moreover, theresults demonstrate that when such pulsed DC are administered incombination with Ab-antigen complexes, that significant enhancement oftotal specific (>35 fold) and relative specific (>10-fold) PSA IgGresponses in SCIDhu PBL mice is obtained (compared to eitherimmunization strategy performed separately). Also, this novelimmunization strategy enhanced human lymphocyte engraftment withoutenhancing XGVHD as in other “enhanced” SCIDhu systems. Moreover, thismethod was reproducible in three separate experiments using differentPBMC donors.

This approach and the technology developed around it is significant asit enables rapid, reproducible production of clinically superiorproducts (human monoclonal antibodies) compared to antibodies based onrodent antibodies). These human monoclonal antibodies are useful forimmunotherapy or immunoprophylaxsis, e.g., treatment or prevention of ofcancer and viral infections.

The described methodology should be useful for generating human MoAbspecific to any relevant target antigen (e.g., Macrophage InhibitoryFactor, E7 antigen, CEA, HIV, etc.). However, being a biological system,it is impossible to predict with absolute certainty the extent ofvariation of the conditions or parameters that will provide optimalresults for different antigens, e.g., the exact number of cells or theexact quantity of Ab-PSA complex that results in optimal antibodyproduction or specificity. However, this can be determined by one ofordinary skill using routine optimization.

The preferred antigens will comprise those expressed by human diseasestreatable by monoclonal antibodies (wherein treatment includestherapeutic and prophylactic therapy), e.g., cancers, parasiticinfections and viral infections. Examples of diseases treatable by humanmonoclonal antibodies include, by way of example, cancers such asbreast, brain, cervical, ovarian, prostate, bladder, pancreatic,myeloma, kidney, colorectal, nasoparingeal, endometrial, lung, liver,leukemia, lymphoma, colon, stomach, skin, among others, viral diseases,including those caused by HIV, hepatitis, papillomavirus, respiratorysyncytial virus, herpes, etc., and parasitic diseases, e.g., malaria.

In the preferred embodiments, the antigen will be selected frommelanocytic differentiation antigens, e.g., gp100 (Kawakami et al, J.Immunol., 154:3961-3968 (1995); Cox et al, Science, 264:716-719 (1994)),MART-1/Melan A (Kawakami et al, J. Exp. Med., 180:347-352 (1994);Castelli et al, J. Exp. Med., 181:363-368 (1995)), gp75 (TRP-1) (Wang etal, J. Exp. Med., 186:1131-1140 (1996)), and Tyrosinase (Wolfel et al,Eur. J. Immunol., 24:759-764 (1994); Topalian et al, J. Exp. Med.,183:1965-1971 (1996)); melanoma proteoglycan (Hellstrom et al, J.Immunol., 130:1467-1472 (1983); Ross et al, Arch. Biochem Biophys.,225:370-383 (1983)); tumor-specific, widely shared antigens, e.g.,antigens of MAGE family, for example, MAGE-1, 2, 3, 4, 6 and 12 (Van derBruggen et al, Science, 254:1643-1647 (1991); Rogner et al, Genomics,29:729-731 (1995)), antigens of BAGE family (Boel et al, Immunity,2:167-175 (1995)), antigens of GAGE family, for example, GAGE-1, 2 (Vanden Eynde et al, J. Exp. Med., 182:689-698 (1995)), antigens of RAGEfamily, for example, RAGE-1 (Gaugler et al, Immunogenetics, 44:323-330(1996)), N-acetylglucosaminyltransferase-V (Guilloux et al, J. Exp.Med., 183:1173-1183 (1996)), and p15 (Robbins et al, J. Immunol.,154:5944-5950 (1995)); tumor specific mutated antigens; mutatedβ-catenin (Robbins et al, J. Exp. Med., 183:1185-1192 (1996)), mutatedMUM-1 (Coulie et al, Proc. Natl. Acad. Sci. USA, 92:7976-7980 (1995)),and mutated cyclin dependent kinases-4 (CDK4) (Wolfel et al, Science,269:1281-1284 (1995)); mutated oncogene products: p21 ras (Fossum et al,Int. J. Cancer, 56:40-45 (1994)), BCR-abl (Bocchia et al, Blood,85:2680-2684 (1995)), p53 (Theobald et al, Proc. Natl. Acad. Sci. USA,92:11993-11997 (1995)), and p185 (HER2/neu (Fisk et al, J. Exp. Med.,181:2109-2117 (1995)); Peoples et al, Proc. Natl. Acad. Sci. USA,92:432-436 (1995)); mutated epidermal growth factor receptor (EGFR)(Fugimoto et al, Eur. J. Gynecol. Oncol., 16:40-47 (19965)); Harris etal, Breast Cancer Res. Treat, 29:1-2 (1994)); carcinoembryonic antigens(CEA) (Kwong et al, J. Natl. Cancer Inst., 85:982-990 (1995)); carcinomaassociated mutated mucins, for example, MUC-1 gene products (Jerome etal, J. Immunol., 151:1654-1662 (1993), Ioannides et al, J. Immunol.,151:3693-3703 (1993), Takahashi et al, J. Immunol., 153:2102-2109(1994)); EBNA gene products of EBV, for example, EBNA-1 gene product(Rickinson et al, Cancer Surveys, 13:53-80 (1992)); E7, E6 proteins ofhuman papillomavirus (Ressing et al, J. Immunol., 154:5934-5943 (1995));prostate specific antigens (PSA) (Xue et al, The Prostate, 30:70-78(1997)); prostate specific membrane antigen (PSMA) (Israeli et al,Cancer Res., 54:1807-1811 (1994)); PCTA-1 (Sue et al, Proc. Natl. Acad.Sci. USA, 93:7252-7257 (1996)); idiotypic epitopes or antigens, forexample, immunoglobulin idiotypes or T cell receptor idiotypes (Chen etal, J. Immunol., 153:4775-4787 (1994); Syrengelas et al, Nat. Med.,2:1038-1040 (1996)).

The antigen will preferably be administered to a SCID mouse in the formof an antigen-antibody complex as described supra. Also, as describedsupra, the antigen or more preferably antigen-antibody complex will beused for in vitro priming of autologous dendritic cells, e.g.,autologous peripheral blood dendritic cells. The amount and duration ofsuch in vitro priming will be that which results in an enhancement ofhuman antibody production, when the resultant primed dendritic cells areused as immunizing agents in SCID mice. As disclosed, preferably SCIDmice will be immunized with autologous dendritic cells which have beenpulsed in vitro with an antigen-antibody complex and further immunizedwith such antigen-antibody complex as this has been shown to confersynergistic benefits (enhance total antisera-specific antibody responseand relative specific IgG antibody response).

Also, it is desirable that EBV transformation be effected duringimmunization. After immunization, human antibody secreting cells will beisolated from such SCID mice and used to clone human monoclonalantibodies. This may be effected by known methods.

Monoclonal antibodies possessing desirable properties (minimum antigenbinding affinity and avidity) obtained by such methods are useful ashuman therapeutics and prophylactics. These human monoclonal antibodieswill be administered by known methods, e.g., systemically orparenterally, e.g., orally, subcutaneously, intravenously,intramusculatory, topically, by infusion, to patients in need of suchtreatment.

The administered dosage will be a dosage that results in therapeutic orprophylactic benefits. Generally, such dosage will range from about0.001 to 100 mg/kg, more preferably 0.01 to 50 mg/kg, still morepreferably 0.1 to 5 mg/kg body weight. Moreover, such dosage will varydependent upon the condition of the patient, the disease condition,whether other therapies are also being effected, among other factors.

Typically, the antibody will be administered in combination with apharmaceutically acceptable carrier or excipient, e.g., phosphatebuffered saline, optionally in combination with adjuvants that enhancehumoral or CTL immunity.

In the case of prostate specific antigen specific antibodies, theseantibodies will be used for the treatment or prevention of prostatecancer as this is a known antigen expressed during prostate cancer.

1. A method for producing human antibodies in SCID mice which comprisesimmunizing SCID mice with dendritic cells which have been contacted(pulsed) in vitro with an antigen-antibody complex.
 2. The method ofclaim 1, wherein the antigen is prostrate specific antigen.
 3. Themethod of claim 2, wherein the antibody is a mouse IgG_(2a) antibody. 4.The method of claim 1, wherein the dendritic cells comprise autologousperipheral blood dendritic cells.
 5. The method of claim 1, whichincludes EBV transformation during the immunization step.
 6. The methodof claim 1, wherein the antibody is anti-PSA IgG₂, monoclonal antibodyand the antigen is PSA.
 7. The method of claim 1, wherein said SCID miceare immunized with antigen-antibody complex prior to a immunization withdendritic which have been contacted (pulsed) with antigen-antibodycomplex.
 8. The method of claim 1, wherein the second immunization iseffected about 1 to 15 days after the first.
 9. The method of claim 8,wherein said second immunization is effected about 7 days after thefirst immunization.
 10. The method of claim 7, which further includes atleast one additional immunization (“boosting”) wherein SCID mice isadministered antigen or antigen-antibody complex.
 11. The method ofclaim 10, wherein a boosting step is effected about a week after thefirst immunization.
 12. The method of claim 11, wherein another boostingstep is effected about two weeks after the first immunization.
 13. Themethod of claim 12, wherein another boosting step is effected aboutthree weeks after the first immunization.